According to Toshihide Kobayashi, Chief Scientist at the RIKEN Advanced Science Institute, “People think cholesterol is bad for health. But without cholesterol, we could not survive. Cholesterol is important, but its function remains elusive.” He and his colleagues are exploring the function of cholesterol by using their unique method for visualizing it in the cell, and are working to develop drugs that suppress the secretion of ‘bad cholesterol’ with the aim of fighting against metabolic syndrome.

METABOLIC SYNDROME AND CHOLESTEROL

A person whose abdominal circumference at the level of the navel is 85 cm or more (for men) or 90 cm or more (for women) may be suffering from visceral fat type obesity, a condition characterized by fat accumulation in the internal organs. If a person has two or more out of hyperglycemia, hypertension, and lipid abnormality, in addition to this type of obesity, he or she is diagnosed with metabolic syndrome (visceral fat syndrome). Metabolic syndrome is dangerous because of its association with life-threatening diseases such as heart disease and cerebral stroke. The condition is likely to result in the deposition of cholesterol and other lipids on vascular walls and, if left untreated, to lead to the progression of arteriosclerosis.

Cholesterol is biosynthesized in cells, but it is also taken from food. When a large amount of cholesterol is taken from food, the blood cholesterol level rises, which in turn can lead to the progression of arteriosclerosis.

“However, cholesterol is essential for the maintenance of life,” explains Kobayashi. “In the body, cholesterol is produced with the consumption of a great deal of energy. Because cholesterol is of paramount importance to the human body, there is no mechanism for its degradation. Hence, excess cholesterol must be discharged from the body mainly through the excretion of bile salts. The body has long evolved to acquire cholesterol, and it is only in the past few decades of people eating too much food that the intake of cholesterol has become excessive and a problem.”

CHOLESTEROL, A MAJOR COMPONENT OF BIOMEMBRANES

Cholesterol is a type of lipid. After water and protein, lipids are the third most abundant of the substances making up our body. Lipids serve as an energy source in the form of fat, and also form structures such as the cell membrane. As a lipid, cholesterol is one of the major components essential to the construction and maintenance of biomembranes.

The phospholipid molecule, another major component of biomembranes, has two distinct portions: a hydrophilic head (water-compatible) and a hydrophobic tail (water-repelling). Two layers of phospholipid molecules lie with their hydrophobic tails facing inwards, forming a lipid bilayer. The cell membrane comprises cholesterol, lipid bilayers and other forms of lipids.

A lipid bilayer can be made artificially from a single kind of lipid, whereas biomembranes are known to comprise several thousand kinds of lipids. Biomembranes made of lipids with different physical properties lose integrity and collapse in the absence of cholesterol; the body is therefore unable to survive without it.

However, little is known about the functions of cholesterol other than maintaining biomembranes. This is because it has been difficult to follow the fate of cholesterol and other lipids as a result of their smaller molecular size than that of proteins.

CHOLESTEROL CONCENTRATIONS ARE CRITICAL TO BIOMEMBRANE FUNCTIONS

Why are thousands of kinds of lipids required to produce biomembranes? To answer this question, the ‘lipid raft’ was hypothesized by Kai Simons and colleagues in Germany around 1988.

Cells cannot function normally without exchanging information and material with the outside. Lipid bilayers themselves do not allow an exchange of information and material, however, protein receptors embedded in lipid bilayers perform this function.

Information received by a receptor from outside the cell is transmitted to a target protein in the cell. At that time, the process of information transmission is complete. However, because the receptor and this specific intracellular protein are not always present at fixed places in the membrane, it is thought that the efficiency of information transmission is increased when the receptor and the intracellular protein are mobilized to a particular region depending on the information. In biomembranes, different kinds of lipids gather to form different characteristic regions that attract particular proteins. In this way a wide variety of patterns of information transmission and material migration is achieved efficiently. This is why thousands of kinds of lipids are required to form biomembranes. In view of these processes, Simons and others named one of these regions a ‘lipid raft’ and predicted that it comprises sphingolipids, eukaryotic plasma membrane lipids that form solid membrane, and cholesterol gathering at one place.

When the lipid raft theory was proposed by Simons, Kobayashi was studying under his tutelage. “The most important thing in science is to establish a hypothesis that can be tested. Even if the hypothesis proves false, science can evolve by testing the hypothesis. I learned this principle from Simons. This concept is seldom considered in Japanese biological research, where emphasis is placed on discovering facts. Of course this attitude is important, but drawing up a hypothesis is also important.”

Does the lipid raft really exist? This question still has not been fully answered, even now, 20 years after the hypothesis was proposed. Kobayashi and his colleagues developed a new technique to reveal sphingolipids and cholesterol, and conducted experiments to test the hypothesis.

“The results were unexpected. We found a region where sphingolipids are present at high density, as predicted by the lipid raft hypothesis. When we looked at cholesterol, however, it was widely distributed in places other than the sphingolipid region.”

Kobayashi and his colleagues found that cholesterol is distributed much more widely than in the region of sphingolipids predicted by the lipid raft hypothesis. However, testing the hypothesis represented scientific advance.

“By studying cholesterol, we found that the distribution of its concentration has a key role in the function of biomembranes. For example, we discovered that a certain type of information transmission is suppressed by membrane domains with a high cholesterol concentration, whereas information is fully transmitted when the cholesterol is diluted.”

Receptors that stop specific information transmission gather at areas with high cholesterol concentrations to block the transfer efficiently. Without such high-cholesterol concentration areas, efficient blocking is impossible and information is transmitted. “Another possibility is that some receptors may gather at areas with higher cholesterol concentrations, and others at areas with lower cholesterol concentrations.” (Fig. 1)

Thus the concentration of the unique lipid cholesterol might be able to control the efficiency of information transmission through the mobilization of membrane receptors.

If cholesterol is itself capable of efficiently achieving a wide variety of patterns of information transmission, thousands of kinds of lipids do not seem necessary. In that case, why are thousands of lipids required? “Nobody knows the answer. We have only just begun to elucidate the function of a single lipid called cholesterol. The functions of thousands of other lipids remain to be clarified. We also need to understand the functions of multiple kinds of lipids in combination. There are so many things to be explained – this is the state of lipid research today. I now recognize that lipid research is indeed difficult.”

CHALLENGING METABOLIC SYNDROME

At the Lipid Biology Laboratory, researchers are conducting an intensive study to reveal cholesterol and elucidate its functions.

“We are the only laboratory that is able to watch the distribution of cholesterol in cells in detail (Fig. 2). The functions of cholesterol are now being unveiled one after another.”

Kobayashi and his colleagues found that cholesterol also has a key role in the process of cell division. “At low concentrations of cholesterol in the cell membrane, cell division ceases at a particular stage. We want to examine how cholesterol is involved in cell division.”

The importance of cholesterol was also demonstrated in endocytosis, the phenomenon in which substances from the extracellular environment are internalized into cells, along with part of their own membrane. “The rate of a certain type of endocytosis varies widely depending on cell density. It was found that in this process, the cell cholesterol content changes dramatically. As the cell density increases, the intracellular cholesterol content increases and the rate of endocytosis decreases (Fig. 3).”

In addition to working on the functions of cholesterol in basic biological phenomena such as cell division and endocytosis, Kobayashi and his colleagues are engaged in research to fight against metabolic syndrome. They are focusing on the cholesterol that is present in a cell organelle known as the endoplasmic reticulum.

In our body, cholesterol is biosynthesized mainly in the endoplasmic reticulum by hepatocytes; in its turn, cholesterol binds to a certain protein and migrates to the cell membrane, from which it is secreted into the blood as LDL, lipoprotein particles named low-density lipoprotein. Cells can incorporate the LDL and make use of the cholesterol contained in these particles.

However, if the blood concentration of cholesterol derived from LDL and food is persistently high, the cholesterol will accumulate on the vascular walls, possibly causing arteriosclerosis. This is the reason why LDL is sometimes referred to as “bad cholesterol.”

A compound that lowers cholesterol concentrations in hepatocytes was developed, and it was expected to be used as a prophylaxis for arteriosclerosis by reducing the amount of secreted LDL. However, it was found that the compound alone is ineffective in decreasing the secretion of LDL.

“We have discovered that the cholesterol concentration in the endoplasmic reticulum of hepatocytes controls the secretion of LDL. If we can clarify the underlying mechanism at the molecular level, it will be a significant contribution to the development of a drug that decreases the amount of secreted LDL.”

Kobayashi and his colleagues are conducting joint research with France’s Institut National de la Santé et de la Recherche Médicale (INSERM). “In the near future I want to obtain results that will help develop a drug that reduces the amount of secreted LDL.”

TOWARDS A NEW STAGE OF LIPID RESEARCH

Kobayashi and his colleagues have explored unknown functions of lipids by making them visible. To watch a particular lipid such as cholesterol selectively, it is necessary to develop a protein that binds to the target lipid only, and then use it as a marker. However, suitable proteins are currently larger than the lipid molecules under study. For this reason, experiments must take into account the possible influence of the marker protein on lipid functions.

“Of course, the best way will be to watch lipids without marking them. We are working on developing a method to do this in collaboration with physicists and chemists at RIKEN,” says Kobayashi.

The Lipid Biology Laboratory is planning to expand its joint research projects. “Of various biomolecules, DNA, protein, and sugar chains have been investigated in detail, but further work is required on lipids. With a focus on lipids, we want to conduct joint research involving researchers in a broad range of fields, including not only biology, but also physics and chemistry, both in RIKEN and elsewhere. Many diseases besides arteriosclerosis are caused by lipids. We would like to cooperate with researchers in medical sciences in characterizing and fighting against diseases caused by lipids.”

A new era of lipid research is about to start at RIKEN.

About the researcher

Toshihide Kobayashi was born in Tokyo, Japan, in 1956. He graduated from the Department of Applied Chemistry, Waseda University, in 1978, and obtained his PhD in 1983 from the Faculty of Pharmaceutical Sciences, the University of Tokyo. After a period in the USA (Carnegie Institution of Washington, Fogarty Fellow), Germany (European Molecular Biology Laboratory, Human Frontier Science Program Fellow) and Switzerland (University of Geneva, Maître-assistant), he returned to Japan in 1999 as a team leader of the Sphingolipid Functions Laboratory, Supra-Biomolecular System Research Group, RIKEN Frontier Research System. In 2003, he was promoted to the position of chief scientist of the Lipid Biology Laboratory, Discovery Research Institute of RIKEN. His research focuses on elucidating the function of lipids through revealing the molecular organization and function of lipids and lipid domains in biomembranes.